The long-term goal of this project is to define the role of the ceramide metabolizing enzyme family of ceramidases in inflammation and to target these enzymes for novel anti-inflammatory therapy. The PI's laboratory has an established track record of expertise in sphingolipid metabolism and function. Studies from the previous funding period have led us into a novel exciting direction on the role and regulation of ceramidases in and their metabolic products in inflammation. Ceramidases breakdown ceramide to generate sphingosine and sphingosine-1-phosphate (S1P) which in turn mediates several biologic activities, including inflammatory responses. In this competing renewal we have compelling new data, whereby we have implicated this pathway as a key regulator of cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production. In addition we find that at least one ceramidase is involved in the TNF inflammatory response. This proposal will therefore, test the hypothesis that ceramidases regulate cytokine-mediated chronic inflammation, and that inhibiting this ceramidase activity inhibits inflammatory responses. To test this hypothesis we propose the following aims: 1) Determine which ceramidases are regulated in inflammation and determine the mechanisms of this regulation. This will be done by determining which of the ceramidases (acid, neutral and alkaline) is/are upregulated in inflammation and studying the mechanisms of this regulation in cell models of inflammation, and by determining the expression and cell-type distribution of ceramidases in inflammatory tissues from humans and from animal models of inflammation. 2) Establish the function of ceramidases in regulation of inflammation and determine the mechanisms of action of their products. This will be done by demonstrating that ceramidases and their metabolic products have a significant role in regulating inflammation by evaluating the effect of over expression of ceramidases in cells and in vivo models of inflammation, and determining the mechanisms by which ceramidases regulate inflammatory pathways (S1P, NF-KB, ERKs, COX-2). 3) Determine the relative contribution of each of the ceramidases in inflammation and dissect the mechanisms involved. This will be done by blocking ceramidase activity in cells and in vivo using small interfering RNA to the different ceramidases. In addition we will test different compounds that we synthesized for their ability to inhibit ceramidases in cells and in vivo models of inflammation. We will also test if ceramidase K/O mice are protected from inflammation in a model of IBD colitis and TNF-induced arthritis. These studies will enable us to gain important insight into the role of the ceramidase pathway in inflammatory responses and may also provide novel therapeutic approaches to inflammation. PUBLIC HEALTH RELEVANCE: NARRATIVE. Relevance to Veterans Health. The VA patient population has a high incidence of chronic inflammatory conditions, in particular, arthritis and colitis. These conditions are difficult to treat and generally are not sensitive to many available modalities of treatment. Moreover, chronic inflammatory conditions are increasingly thought to lead to cancer. Tumor necrosis factor (TNF) and COX-2 are implicated in most of these chronic inflammatory conditions and recent highly effective anti-inflammatory therapy is geared at blocking their action. However, these anti-inflammatory agents have proven to have cardiovascular and other side effects. Our preliminary studies demonstrate that sphingolipid molecules are intermediates in the action of TNF on COX-2. We will therefore utilize models of rheumatoid arthritis and inflammatory colitis to study the sphingolipid regulating enzymes ceramidases in these models of inflammation and target these enzymes for novel therapies in the treatment of chronic inflammation.